In all kingdoms of life RNAs are modified both during and after synthesis, thereby altering the stability, higher-order structure, and activity of RNA through changes in base stacking and hydrogen bonding. These generally conserved modifications are located at key functional regions and are required for optimal RNA function. Approximately 0.8% of the coding capacity of the E. coli cell is dedicated to enzymes that modify RNA with unique or limited multisite specificity. The aim of this application is to determine the basis for selectivity and catalysis in two of the most abundant families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases) and pseudouridine synthases (synthases). Our reported structures of two m5U MTases have yielded a model for specific recognition in which the RNA substrate is refolded onto the enzyme and a base is flipped out into the active site. This work proposes to test, refine and elaborate that model in kinetic and structural terms. Our to-date and proposed studies of five sub-classes of bacterial (synthases that modify stem loops of tRNAs (TruA and TruB), a stem loop (RluD), and a helix (RluB and RluF)) rRNA is yielding models for site specificity and regional specificity that will serve as paradigms for synthases that play roles in human health. The field will thus be advanced into regulation in human biology with our proposed work on human Pus1 and Pus3, two enzymes that regulate nuclear receptor signaling by pseudouridylating an RNA activator of nuclear receptors (steroid receptor RNA activator, SRA). Defects in these enzymes cause sideroblastic anemia. The strategy for all enzyme studies remains to express key proteins, to define the nature of the substrate as a complete tRNA or smaller stem-loop, and then to determine crystal structures of enzyme-RNA complexes at critical points close to the transition state. Mutations in protein and RNA are used to determine the mechanisms by which the individual target base is brought into the catalytic site. Mechanism is also probed through measures of the rates of individual steps including base-flipping, kcat/Km and Kd determinations. RNA structural changes during the reactions will be monitored through time-resolve fluorescence changes of 2-aminopurine labeled substrates. Targeted molecular dynamics is used to guide experiments. Finally, we seek to determine the structure of a human tRNA adenine MTase as a potential new anti-HIV drug target. It generates an essential modification that controls reverse transcription of HIV RNA. The expression and purification of this two subunit AdoMet dependent RNA methylase is to lead to a structure, and to a screening approach to specific inhibitor discovery. PUBLIC HEALTH RELEVANCE: The aim is to determine the basis for action of two of the most abundant families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases), and pseudouridine synthases (synthases), and to elucidate the roles of two human synthases and one human RNA MTase with importance in human disease. The importance of synthase activity is underlined by disorders such as sideroplastic anemia. The RNA MTase is a potential new drug target for anti-HIV therapy.